The nucleotide (p)ppGpp is critical for stress resistance and antibiotic tolerance. In the Gram-negative bacterium Escherichia coli, (p)ppGpp interacts with RNA polymerase to reprogram transcription upon stress. However, in Firmicutes, a major class of Gram- positive bacteria comprised of pathogens (Staphylococcus, Enterococcus, Listeria, Bacillus anthracis) and beneficial microbes, how (p)ppGpp mediates stress resistance remains poorly understood. The central hypothesis of this proposal is that, in Firmicutes, (p)ppGpp regulates GTP levels to adjust pleiotropic aspects of cellular metabolism to the external environment, thus maintaining homeostasis and facilitating antibiotic tolerance. Our recent work with the bacterium Bacillus subtilis, a widely studied, genetically amenable model for Firmicutes, lends strong support for this hypothesis. Integrating genomic and metabolomic approaches with genetics and biochemistry, we identified enzymes in the GTP biosynthesis pathway as major targets for (p)ppGpp action, and unmasked a novel GTP-(p)ppGpp feedback loop that is critical for cellular viability. Our discoveries thus provide a previously unrecognized view of (p)ppGpp synthesis and function in B. subtilis. We aim to build on this foundation to develop a comprehensive model of stress-management via (p)ppGpp-GTP in B. subtilis that our data predicts will be applicable to Firmicutes and beyond. The specific aims are: 1) Characterize how (p)ppGpp regulates its targets Gmk and HprT; 2) Elucidate how (p)ppGpp controls GTP feedback and glycolysis; and 3) Define how (p)ppGpp contributes to antibiotic tolerance.